Identification and Molecular Cloning of a p21-activated Serine/Threonine Kinase That Is Rapidly Activated by Thrombin in Platelets

doi:10.1074/jbc.270.44.26690

Identification and Molecular Cloning of a p21-activated Serine/Threonine Kinase That Is Rapidly Activated by Thrombin in Platelets (*)

From the (1) Glaxo-IMCB Group, Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore 0511, Singapore and the
(2) Department of Neurochemistry, Institute of Neurology, 1 Wakefield Street, London WC1N 1PJ, United Kingdom

^§ To whom correspondence should be addressed. Tel.: 65-772-6167; Fax: 65-774-0742.

Abstract

The brain-enriched p21-activated serine/threonine kinase, p65, was identified and purified on the basis of overlays with [-P]GTP-Cdc42 onto SDS-fractionated proteins (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L.(1994) Nature 367, 40-46). In this study, the ubiquitously expressed p21 binding protein with relative molecular weight of 62,000 was purified from rat testes and shown to contain peptides related to PAK. It has thus been designated as the -PAK isoform (α- and β-isoforms being brain enriched). Isolation of -PAK cDNAs show that the kinase is highly conserved with α-PAK in both the p21 binding and kinase domains. The purified protein exhibited kinase activity that was activated by GTP-Cdc42 or GTP-Rac1 in vitro. In platelets, a p62 in situ renaturable kinase was recognized by antibodies raised against -PAK. This thrombin-activated protein kinase appears to coprecipitate with another kinase of M_r 86,000, suggesting that PAK may be part of a thrombin-responsive signaling complex.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GAP

GTPase-activating protein

PCR

polymerase chain reaction

GST

glutathione S-transferase

PVDF

polyvinylidene difluoride

PBS

phosphate-buffered saline

MBP

myelin basic protein

MES

morpholinoethanesulfonic acid

kb

kilobase(s)

bp

base pair(s)

PAK

p21-activated kinase

ACK

activated Cdc42-associated kinase

GTPS

guanosine 5′-3-O-(thio)triphosphate.
↵² T. Leung and L. Lim, unpublished data.
- Received July 5, 1995.
- Revision received August 23, 1995.